Catalytic mechanism of active-site serine β-lactamases: role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly triad

Abstract

The role of the conserved hydroxy group of the Lys-Thr(Ser)-Gly [KT(S)G] triad has been studied for a class A and a class C beta-lactamase by site-directed mutagenesis. Surprisingly, the disappearance of this functional group had little impact on the penicillinase activity of both enzymes. The cephalosporinase activity was much more affected for the class A S235A (Ser235-->Ala) and the class C T316V (Thr315-->Val) mutants, but the class C T316A mutant was less impaired. Studies were extended to beta-lactams, where the carboxy group on C-3 of penicillins or C-4 of cephalosporins had been modified. The effects of the mutations were the same on these compounds as on the unmodified regular penicillins and cephalosporins. The results are compared with those obtained with a similar mutant (T299V) of the Streptomyces R61 DD-peptidase. With this enzyme the mutation also affected the interactions with penicillins and severely decreased the peptidase activity. The strict conservation of the hydroxy group on the second residue of the KT(S)G triad is thus much more easy to understand for the DD-peptidase and the penicillin-binding proteins than for beta-lactamases, especially those of class C.

Characterization of the Inhibitor-Resistant SHV β-Lactamase SHV-107 in a Clinical Klebsiella pneumoniae Strain Coproducing GES-7 Enzyme

“…Amoxicillin, with a k cat value of 657 s Ϫ1 , was the best substrate for SHV-107. Likewise, previous enzymatic studies of TEM-1 and its isogenic mutant Ser235Ala showed similar results for penicillins, although the mutant had greatly reduced cephalosporinase activity (15,20). In the SHV ␤-lactamase family, residue Thr235 is also critical for cephalosporinase activity, because its replacement by Ala235 in SHV-107 leads to severe impairment of the catalytic constant against cephalosporins, as observed in TEM-type enzyme (15,20).…”

Avibactam and Class C β-Lactamases: Mechanism of Inhibition, Conservation of the Binding Pocket, and Implications for Resistance

“…Given the conservation of the avibactam binding pocket residues and the importance of these residues in ␤-lactam recognition and catalysis (25)(26)(27)(28)(29), we investigated whether any variations in these might compromise the inhibition potency of avibactam while still allowing hydrolysis of the ␤-lactam drug and thus result in resistance. Spontaneous resistance frequency experiments were carried out in several isolates carrying class C ␤-lactamases, with an initial focus on Enterobacteriaceae spp.…”

Three Decades of β-Lactamase Inhibitors

“…The functional explanation for the conservation of the Lys234-Thr(Ser)235-Gly236 triad is less well understood. Removal of the hydroxyl group from the middle residue has little impact on penicillin hydrolysis in TEM (but more on the catalytic efficiency of cephalosporins), and despite what may be expected from crystal structure alignments, the study of modified ␤-lactams argues against an interaction between the ␤-lactam carboxylate and the Thr(Ser) hydroxyl group (112,181). In SHV, the Thr235Ala substitution lowers MICs of both penicillins and cephalosporins (174).…”